Avian leukemia virus detection kit based on magnetic particle chemiluminescence method and application thereof

Abstract

The invention belongs to the technical field of biological detection, and discloses an avian leukosis virus detection kit based on a magnetic particle chemiluminescence method and application thereof. The kit comprises a liquid A, a liquid B, a liquid C, a liquid D calibration product and a quality control product, the liquid A comprises magnetic particles coated with an avian leukosis virus antibody, and the liquid B comprises an acridinium ester labeled avian leukosis virus antibody. According to the kit disclosed by the invention, the avian leukosis virus antibody is coated on the magnetic particles, and the magnetic particles have a relatively large surface area, so that the antigen capture efficiency is greatly improved, and the detection specificity and sensitivity are enhanced. In addition, acridinium ester is adopted for labeling, the cost is low, enzyme catalysis is not needed, the influence of environment temperature and humidity on the reaction is small, luminescence is stable, the luminescence efficiency is high, background luminescence is low, the signal-to-noise ratio is high, the detection time is short, the detection efficiency and stability are greatly improved, and rapid detection of large-scale samples in production is facilitated.

Description

Technical Field

[0001] The invention relates to the technical field of biological detection, and in particular to an avian leukosis virus detection kit based on a magnetic particle chemiluminescence method and an application thereof. Background Art

[0002] Avian leukosis is caused by avian leukaemia virus (ALV). It is an infectious disease with the main clinical features of slow growth, poor development, decreased egg production, and malignant tumors in tissues and organs. It can be transmitted horizontally or vertically. The occurrence and prevalence of avian leukosis are distributed worldwide, and there is no effective drug and vaccine for prevention and treatment, causing huge economic losses to the poultry industry. Although my country has implemented a strict ALV purification plan, there are still multiple avian leukosis subgroups such as A, B, E, J and K that are prevalent in my country, and they show the characteristics of mixed infection of multiple subgroups, virus mutation, and continuous expansion of hosts. Efficient, accurate and convenient ALV detection methods are of great significance for the prevention, control and purification of the disease.

[0003] The diagnostic methods of avian leukosis in the national standard "Diagnostic Technology of Avian Leukosis" (GB / T 26436-2010) mainly include clinical symptoms and pathological changes, virus isolation, culture and identification, ELISA detection, and fluorescence quantitative PCR amplification of ALV-J. However, in actual clinical applications, the diagnosis of clinical symptoms and pathological changes requires the diagnostician to be experienced, and Marek's disease also has tumors as clinical features, which can easily cause confusion; the fluorescence quantitative PCR method has high requirements for experimental equipment, conditions and the professional quality of operators, and the ELISA detection method is simple to operate and suitable for a large number of sample detection, but the ELISA detection requires multiple incubations and washings, which is time-consuming. In addition, peroxidase is greatly affected by the environment during the substrate catalysis process. Therefore, it is urgent to develop an avian leukosis virus detection method with high sensitivity and specificity, simple operation, rapid reaction, low cost and suitable for large-scale sample detection. Summary of the invention

[0004] In view of the defects in the prior art, the present invention proposes an avian leukosis virus detection kit based on magnetic particle chemiluminescence method and its application.

[0005] The invention provides an avian leukosis virus detection kit based on a magnetic particle chemiluminescence method, comprising liquid A, liquid B, liquid C, liquid D, a calibrator and a quality control product; the liquid A comprises magnetic particles coated with avian leukosis virus antibodies, the concentration of the magnetic particles is 0.3-0.8 mg / ml, and the particle size of the magnetic particles is 0.05-3.0 μm; the liquid B comprises avian leukosis virus antibodies labeled with acridinium ester, and the concentration of the avian leukosis virus antibodies labeled with acridinium ester is 0.2-0.8 μg / ml; the liquid C comprises one or more of p-hydroxycinnamic acid, hydrogen peroxide, urea peroxide and a preservative; the liquid D is an alkaline reagent, and the alkaline reagent comprises one or more of sodium hydroxide and potassium hydroxide.

[0006] Preferably, the concentration of the magnetic particles is 0.5 mg / ml; the concentration of the acridinium ester-labeled avian leukosis virus antibody is 0.5 μg / ml.

[0007] Furthermore, the solution A also includes 0.01-0.1M buffer, 0.1-0.5 vol% preservative, and 0.1-2.0 μg / ml avian leukosis virus antibody.

[0008] Furthermore, the B solution also includes 0.01-0.1M buffer, 0.1-0.5 vol% preservative, and 0.05-0.5 vol% surfactant.

[0009] Furthermore, the calibrator includes calibrator 1 and calibrator 2; calibrator 1 includes a protein buffer and a preservative, and calibrator 2 includes a protein buffer containing an avian leukosis virus antigen and a preservative; the protein is one or more of bovine serum albumin and fetal bovine serum; wherein the mass percentage of bovine serum albumin is 1 wt%, and the mass percentage of fetal bovine serum is 10 wt%.

[0010] Furthermore, the quality control product includes quality control product 1 and quality control product 2; the quality control product 1 includes a protein buffer and a preservative, and the quality control product 2 includes a protein buffer containing an avian leukosis virus antigen and a preservative; the protein is one or more of bovine serum albumin and fetal bovine serum; wherein the mass percentage of bovine serum albumin is 1 wt%, and the mass percentage of fetal bovine serum is 10 wt%. Preferably, the protein is 1 wt% bovine serum albumin.

[0011] Furthermore, the preservative is one or more of PC150 and PC300.

[0012] Preferably, the preservative is PC 300.

[0013] Furthermore, the buffer is one or more of Tris-HCl buffer and phosphate buffer.

[0014] Furthermore, the surfactant is one or more of Tween 20 and Tween 80.

[0015] Preferably, the surfactant is Tween 20.

[0016] Furthermore, the labeling ratio of the acridinium ester-labeled avian leukosis virus antibody to the acridinium ester is 1:(5-30).

[0017] Preferably, the labeling ratio of the acridinium ester-labeled avian leukosis virus antibody to the acridinium ester is 1:10.

[0018] The invention also provides application of the kit in detecting avian leukosis virus.

[0019] In summary, compared with the prior art, the present invention achieves the following technical effects: 1. The ALV virus detection kit described in the present invention can be adapted to a fully automatic chemiluminescence instrument, which is simple to operate, highly sensitive, accurate and stable in results, low in cost, and short in time, and can effectively improve the detection efficiency. It not only provides a quick and convenient method for diagnosing the disease in chicken farms, but is also suitable for the purification monitoring of avian leukosis in the region.

[0020] 2. The kit of the present invention adopts magnetic microparticle chemiluminescence method, which does not require a catalyst, has high luminescence efficiency, low background luminescence, and high signal-to-noise ratio.

[0021] 3. The kit of the present invention coats the avian leukosis virus antibody on magnetic particles, which have a large surface area, thereby greatly improving the antigen capture efficiency and enhancing the specificity and sensitivity of the detection. 4. The present invention is easy to operate, and the reaction time can be shortened to within 15 min, which significantly improves the detection efficiency.

[0022] 5. The reaction system of the present invention improves the utilization rate of antibodies, and the minimum detection limit corresponds to a concentration of 0.7 PFU / mL, which greatly improves the sensitivity and accuracy of detection.

[0023] 6. The kit of the present invention has good repeatability, a coefficient of variation of less than 3%, and higher quality control.

[0024] 7. The kit of the present invention can effectively control operation time, reduce costs, reduce waste emissions, and is more convenient for clinical use. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0026] Figure 1 It is a correlation regression analysis diagram of the test results of samples with known concentrations of the kit of the present invention. DETAILED DESCRIPTION

[0027] In order to enable those skilled in the art to better understand the scheme of the present invention, the technical scheme in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only embodiments of a part of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work should fall within the scope of protection of the present invention.

[0028] In the present invention, 1-ethyl-(3-dimethylaminopropyl)carbodiimide is referred to as EDC; N-hydroxysuccinimide is referred to as NHS; avian leukosis virus is referred to as ALV; bovine serum albumin is referred to as BSA; tris-hydroxymethylaminomethane hydrochloride is referred to as Tris-HCl; and Proclin 300 is referred to as PC300.

[0029] Example 1 Preparation of Liquid A The magnetic particles were washed 3 times with EDC (4 mg / L) buffer and NHS (4 mg / L) buffer, activated for 1 h, ALV antibody was added, coupled for 3 h, then washed once with EDC (4 mg / L) buffer and NHS (4 mg / L) buffer, 1% BSA was added, mixed, and stored at 4°C overnight to block the excess sites on the surface of the magnetic particles. After washing the coupled magnetic particles twice with buffer (containing Tris-HCl 0.05M, NaCl0.1M, PC300 0.1 vol%, pH7.4), the above buffer was added to adjust the concentration of the magnetic particles to 0.5 mg / ml to obtain solution A. It was packaged in a reagent bottle, 4 ml / bottle, and labeled. The above magnetic particles are a composite of Fe2O3 magnetic nanoparticles and organic polymer materials, and the particle size of the magnetic particles is 0.05-3.0 μm.

[0030] Example 2 Preparation of Solution B Add ALV antibody to 0.01 M PBS buffer, mix thoroughly, and prepare an antibody solution with a concentration of 2 mg / ml; dissolve 2 mg of acridinium ester in 1 ml of DMSO to prepare a concentration of 2 mg / ml. Mix ALV antibody and acridinium ester at a ratio of 1:10, mix immediately, and incubate in a 25°C incubator in the dark for 2 h; add 1 M glycine buffer to react for 1 h, purify and collect antibodies, and dilute the antibodies to a final concentration of 0.5 μg / ml with 0.01 M Tris-HCl buffer containing 0.1 vol% PC300 and 0.1 vol% Tween 20 to obtain solution B. Package in a reagent bottle, 10 ml / bottle, and label well.

[0031] Example 3 Preparation of Solution C, Solution D, Calibrators and Quality Controls Preparation of reagent C: 50 mmol / L hydrogen peroxide, add PC 300 to make the final concentration 0.1%.

[0032] Preparation of reagent D: Add 20g sodium hydroxide and 5.7g hexadecyltrimethylammonium bromide to 200ml purified water, stir to dissolve, add 2.5ml Triton X-100, stir evenly, and add purified water to make up to 1L. Pack quantitatively and store at 2℃~8℃.

[0033] Preparation of calibrator 1: 0.01 M pH 7.4 Tris-HCl buffer was added with BSA and PC300 to a final concentration of 5 wt% and 0.1 vol%, respectively.

[0034] Preparation of calibrator 2: ALV antigen was diluted with 0.01 M Tris-HCl buffer containing 5 wt% BSA and 0.1 vol% PC300 to a final concentration of 200.00 pg / ml.

[0035] Preparation of quality control product 1: 0.01 M pH7.4 Tris-HCl buffer, add BSA and PC300 to make the final concentrations of 5 wt% and 0.1 vol%, respectively.

[0036] Preparation of quality control 2: Dilute ALV antigen with 0.01 M Tris-HCl buffer containing 5 wt% BSA and 0.1 vol% PC300 to a final concentration of 500.00 pg / ml.

[0037] The reagents were prepared according to Examples 1-3 to form a kit, and calibration samples were measured, cut-off values ​​were calculated, and samples were tested on a fully automatic chemiluminescence instrument according to the instrument operating instructions.

[0038] Example 4 Kit Sensitivity Test ALV antigen was diluted with 0.02 M pH7.4 PBS to eight gradient concentrations of 0.00 PFU / mL, 0.7 PFU / mL, 1.75 PFU / mL, 3.5 PFU / mL, 10.5 PFU / mL, 35 PFU / mL, 70 PFU / mL, and 280 PFU / mL as test samples. The reagent kit was prepared using the reagents described in Examples 1-3, and each sample was tested 3 times to calculate the average value. At the same time, the samples were tested in parallel using two brands of commercially available ELISA kits. The results are shown in Table 1. The minimum amount of virus detected by the commercial ELISA kit is 10.5 PFU / mL, while the minimum amount of virus detected by the kit of the present invention can reach 0.7 PFU / mL, which is much higher than the ELISA kit.

[0039] Table 1 Kit sensitivity test results Note: N stands for negative; P stands for positive Example 5 Kit Linearity Test The avian leukosis virus antigen quality control product [VIP (P) 10078] was diluted in a gradient manner and numbered S0 to S7. Each sample was repeated 3 times and the average value was taken. The test data and the standard concentration value were fitted with a least squares method, and the linear correlation coefficient was calculated. The test results of the method are shown in Table 2. The correlation regression analysis between the known concentration and the measured concentration is shown in Table 2. Figure 1 shown.

[0040] Table 2 Test results The results in Table 2 show that the measured results are basically consistent with the known concentrations. Figure 1 The results show that the correlation coefficient between the known concentration and the measured concentration is r=0.9993, which is greater than 0.9900. The above results show that the reagent has good accuracy and specificity.

[0041] Example 6 Test kit precision test A high concentration sample (280 PFU / mL), a medium concentration sample (140 PFU / mL) and a low concentration sample (3.5 PFU / mL) were prepared using the avian leukosis virus antigen quality control product [VIP (P) 10078]. The kits assembled with the reagents prepared using the methods described in Examples 1-3 were tested, and each sample was repeated 10 times to calculate the average value and standard deviation. According to the formula: coefficient of variation CV = (standard deviation / average value) * 100%, the coefficient of variation was calculated to evaluate the precision of the kit, and the results are shown in Table 3. It can be seen from the test results that the coefficient of variation CV of the kit prepared by the present invention is <3%, which has good repeatability and is much lower than the coefficient of variation of the ELISA kit (CV <10%).

[0042] Table 3 Test kit precision test Example 7 Kit Stability Test Reagents were prepared according to the method described in Examples 1-3 to form several kits from the same batch, which were placed in a temperature environment of 37°C and 2-8°C for 0 days, 7 days, 14 days, 28 days, 60 days, and 180 days, respectively, and standards of different concentrations were tested and compared with the results of day 0. The deviation was calculated according to the following formula: Deviation rate = (test result of day N - test result of day 0) / test result of day 0 * 100%, and the test results are shown in Tables 4 and 5. It can be seen from the test results that the test results of the kit of the present invention are less than 5% within 14 days under the storage condition of 37°C, which is relatively stable, and the deviation rate is greater than 5% within 28 days. Under the storage condition of 2-8°C, the deviation rate of the test results within 180 is less than 5%, which is relatively stable, indicating that the kit can be stored for a long time under the condition of 2-8°C.

[0043] Table 4 Stability test of the kit under 37℃ storage condition Table 5 Stability test of the test kit under storage conditions of 2-8℃ The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A kit for detecting avian leukosis virus based on magnetic particle chemiluminescence method, characterized in that: Including solution A, solution B, solution C, solution D, calibration products and quality control products; The A solution comprises magnetic particles coated with avian leukosis virus antibodies, the concentration of the magnetic particles is 0.3-0.8 mg / ml, and the particle size of the magnetic particles is 0.05-3.0 μm. The B solution includes an avian leukosis virus antibody labeled with an acridinium ester, and the concentration of the avian leukosis virus antibody labeled with an acridinium ester is 0.2-0.8 μg / ml; The C liquid includes one or more of p-hydroxycinnamic acid, hydrogen peroxide, urea peroxide, and a preservative. The D solution is an alkaline reagent, and the alkaline reagent includes one or more of sodium hydroxide and potassium hydroxide.

2. The kit according to claim 1, characterized in that The A solution also includes 0.01-0.1M buffer, 0.1-0.5 vol% preservative, and 0.1-2.0 μg / ml avian leukosis virus antibody.

3. The kit according to claim 1, characterized in that The B solution also includes 0.01-0.1M buffer, 0.1-0.5 vol% preservative, and 0.05-0.5 vol% surfactant.

4. The kit according to claim 1, characterized in that The calibrator includes calibrator 1 and calibrator 2; the calibrator 1 includes a protein buffer and a preservative, and the calibrator 2 includes a protein buffer containing an avian leukosis virus antigen and a preservative; the protein is one or more of bovine serum albumin and fetal bovine serum.

5. The kit according to claim 1, characterized in that The quality control products include quality control product 1 and quality control product 2; the quality control product 1 includes a protein buffer and a preservative, and the quality control product 2 includes a protein buffer containing an avian leukosis virus antigen and a preservative; the protein is one or more of bovine serum albumin and fetal bovine serum.

6. The kit according to claims 1-5, characterized in that The preservative is one or more of PC150 and PC300.

7. The kit according to claims 2-5, characterized in that The buffer is one or more of Tris-HCl buffer and phosphate buffer.

8. The kit according to claim 3, characterized in that The surfactant is one or more of Tween 20 and Tween 80.

9. The kit according to claim 1, characterized in that The labeling ratio of the acridinium ester labeled avian leukosis virus antibody to the acridinium ester is 1:(5-30).

10. Use of the kit according to claims 1-9 in detecting avian leukosis virus.

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